Experiment Goals

The ultimate goal of this research is to create a novel biological system that ensures that the demineralization threshold is never crossed in the oral biome. In order to do this, one of the native bacteria, Veillonella parvula, would have to be engineered to: (1) detect and respond to pH drops due to lactic acid and (2) catabolize lactic acid in order to negate a drop in pH. Since V. parvula naturally contains lactic acid catabolic pathways, the detection and response aspect of this system will be the scope of this project. Our experiment has two intermediate goals: (1)Develop a new pH-responsive promoter and (2) construct a dual-state promoter with this new promoter.

The standardization inherent to the synthetic biology field means that genetic manipulation of a model system can in theory be easily translated to other species with the same genetic framework. Since the genome of Escherichia coli is well characterized, it will be used as the model system for our experiment.

This technology has the potential to dramatically change the oral health industry, but it also has applications to any biological system where pH monitoring would be necessary, including water sampling, food processing, and many other industries.

Strains and Media

scherichia coli Top10 (Invitrogen) was used for all transformations and assays. Media included SOB for transformations and LB for overnight cultures. Transformed strains were grown at 37°C using Ampicillin resistance. Primers for PCR were purchased from Integrated DNA Technologies (IDT) and New England Biolabs (NEB) donated all of the restriction enzymes. All sequencing was conducted by the Northwestern Genomics Core.

Forming a Library of Constructs

e low copy plasmid pSB4A5 will be used for the different promoter constructs. The asr and gadA promoters were extracted from the E. coli genome via colony polymerase chain reaction (PCR). The constitutive promoters TacI and Lpp were amplified from pDAK1 and pDAK2 donated by the Jewett Lab6,7. The restriction enzyme cut sites EcoR1, Pst1, Spe1, and Xba1 were used in ligation to create the different constructs (Figure 3). When multiple parts were connected a mixed site was formed between Spe1 and Xba1, which cannot be cut, by any of the restriction enzymes. This leads to the benefit of not having a restriction site in the middle of the construct, and furthermore these standard restriction enzymes can always be used with the final dual-state promoter constructs.

Fluorescence Assays

orescence assays of GFP expression will be conducted to analyze the activity of both the single promoters and the dual-state combinations. This will done by using a plate reader to measure the fluorescence per optical density of cells in minimal media with pH ranging from 7.5 – 3. The reason optical density will be taken into account is to control for fluorescence differences that may be based on different cell counts across trials. Instead the chosen measurement will be analogous to fluorescence per cell, giving much greater insight into the activity of the dual-state promoter in each cell at each pH level.

Results